Method for controlling undesirable byproducts formation caused by contaminating organisms in the production of ethanol from syngas

ABSTRACT

A method of operating a fermentation zone for the production of ethanol from syngas uses a crotonate derivative to prevent or reverse the effects of butyrigen contamination. The crotonate compound works in continuous fermentation processes to reduce or eliminate contamination from butyrate and butanol in the syngas derived ethanol product.

FIELD OF THE INVENTION

This invention pertains to processes for the low energy, anaerobicbioconversion of hydrogen and carbon monoxide in a gaseous substratestream to oxygenated C₂ compounds such as ethanol by contact withmicroorganisms in a fermentation system with high conversion efficiencyof both hydrogen and carbon monoxide. The method of this inventionreduces production of undesirable byproducts such as butyric acid,butanol and other longer chain organic acids or alcohols that resultfrom bacterial contaminants in the fermentation system.

BACKGROUND

Bioethanol production for use as a liquid motor fuel is increasingworldwide. Such biofuels include, for example, ethanol that can beblended with gasoline with a wide range of compositions. One of themajor drivers for bioethanol is its derivation from renewable resourcesby fermentation and bioprocess technology. Conventionally, biofuels aremade from readily fermentable carbohydrates such as sugars and starches.For example, the two primary agricultural crops that are used forconventional bioethanol production are sugarcane (Brazil and othertropical countries) and corn or maize (U.S. and other temperatecountries). The availability of agricultural feedstocks that providereadily fermentable carbohydrates is limited because of competition withfood and feed production, arable land usage, water availability, andother factors. Consequently, lignocellulosic feedstocks such as forestresidues, trees from plantations, straws, grasses and other agriculturalresidues are looked to as feedstocks for biofuel production. Unlikeutilization of fossil fuels, deriving bioethanol from such plant or evenmunicipal waste sources provides an environmentally sustainable resourcefor the production of liquid fuels.

A highly efficient route to the production of bioethanol is thegasification of biomass or other organic matter into a substrate gascomprising CO and/or hydrogen followed by the conversion of the gas toethanol using homoacetogenic microorganisms. Methods for such conversionare known from U.S. Pat. No. 7,285,402 B2, US 20110059499 A1, US20090215163 A1, and others.

Typically the substrate gas for carbon monoxide or hydrogen conversionsis derived from a synthesis gas (singes) from the gasification ofcarbonaceous materials, reforming of natural gas and/or biogas fromanaerobic fermentors or from off streams of various industrial methods.The gas substrate contains carbon monoxide, hydrogen, and carbon dioxideand usually contains other components such as water vapor, nitrogen,methane, ammonia, hydrogen sulfide and the like. (For purposes herein,all gas compositions are reported on a dry basis unless otherwise statedor clear from the context.)

Production of ethanol from the substrate gas by these methods requiressignificant amounts of hydrogen and carbon monoxide. For instance, thetheoretical equations for the conversion of carbon monoxide and hydrogento ethanol are:6CO+3H₂O.C₂H₅OH+4CO₂6H₂+2CO₂.C₂H₅OH+3H₂O

As can be seen, the conversion of carbon monoxide results in thegeneration of carbon dioxide. The conversion of hydrogen involves theconsumption of hydrogen and carbon dioxide, and this conversion issometimes referred to as the H₂/CO₂ conversion. For purposes herein, itis referred to as the hydrogen conversion.

Syngas fermentation processes suffer from the poor solubility of the gassubstrate, i.e., carbon monoxide and hydrogen, in the liquid phase ofthe fermentation menstruum. Munasinghe, et al., in Biomass-derivedSyngas Fermentation in Biofuels: Opportunities and Challenges, BiosourceTechnology, 101 (2010) 5013-5022, summarize volumetric mass transfercoefficients to fermentation media reported in the literature for syngasand carbon monoxide in various reactor configurations and hydrodynamicconditions. As a result biofermentation processes for the production ofethanol will require large volumes of fermentation liquid. For examplecommercial scale plants, those with capacities of 55 million gallons ormore, will require fermentation zones that utilize vessels holding amillion gallons or more of the fermentation liquid.

To maintain the efficiency of producing ethanol by such fermentationzones there is a need to maximize the production of C₂ oxygenatedproducts while minimizing the production of higher carbon chain productssuch as C₄, C₆, C₈ and higher organic acids or alcohols. The knownmethods seek to accomplish this efficiency by utilizing homoacetogenicbacteria that have a very high degree of selectivity for the productionof C₂ products. By their nature the homoacetogenic organisms thatconvert the gas substrate to ethanol do not have the pathways to makethese longer carbon chain products.

The ability of homoacetogenic organisms to survive with minimal media onthe CO and H₂ substrate under anaerobic condition provides a protectionagainst many biological contaminants that require much differentenvironments. However, the size and scale of the fermentation zones andoverall facilities necessary for the production of ethanol on acommercial basis precludes axenic operation of the facilities. As aresult microbial contamination will inevitably occur at some point andcan degrade the production by producing such higher chain byproductsthat can result in 20 percent or more of byproducts and thus severelyreducing the yield of ethanol, or other desirable products.

While there are many potential contaminants, one class of potentiallycommon contamination will produce butyric acid, butanol and other longerchain organic acids or alcohols. Microorganisms that produce suchcompounds as part of their primary metabolism are referred to asbutyrigens. There are many classes of butyrigens. A major class utilizescarbohydrates and other carbon compounds such as amino acids, lipids,etc. Another class of butyrigens uses syngas and yet another class ofbutyrigens can utilize ethanol and acetate with transferase enzymepathways. Since, for the reasons previously mentioned, it is notpossible to operate such large fermentation axenically, all of thesebutyrigen contamination sources will exist.

Once the butyrigen contamination takes hold in a large scalefermentation vessel it can destroy the commercial viability of theprocess by shifting feed conversion from desired products and makingproduct recovery impractical. Designing product recovery facilities forwide variations in composition and concentration of liquid compoundswill add prohibitive cost. The large volume of the fermentation liquidand the time to incubate the microorganisms to production concentrationsmake flushing and restarting of the facility impractical as well.

Therefore methods are sought to eliminate or inhibit the growth ofbutyrigens in a large scale fermentation zone without disrupting theongoing production of ethanol or other products such as acetic acid,propanol, or propionic acid from such a fermentation zone.

SUMMARY

By this invention a class of crotonate compounds have been found toinhibit the growth of a butyrigen population while not disrupting theproductivity of light oxygenate products such as ethanol, acetic acid,propanol, and propionic acid by homoacetogenic or heteroacetogenicmicroorganisms. Achieving this discovery required the identificationthat these compounds act as bacteriostatic or bacteriocidal agents tothe butyrigens while not inhibiting the growth of the homoacetogens.Whether the compound acts as a bactericide or bacteriostatic agent, itsability to act in vivo is equally important for its effectiveness inpreserving production of desired products in a large scale fermentationzone. The discovered class of compounds was found to be effective invivo and therefore will act to inhibit or retard butyrigen contaminationwithin the fermentation vessel and may be introduced as an additive inthe process as needed. Crotonate derivatives that act in bacteriocidalor bacteriostatic manner of this invention are referred to herein asbutyl retardants.

Effective crotonate compounds include di-halogen substituted crotonatecompounds. The di-halogen substituted crotonate compounds include acidand esters. Specific compounds include Ethyl 4,4,4, Triflouromethyl3(trifluromethyl)crotonate, 4,4,4,trifluoro 3-(triflouromethyl)crotonicacid, Ethyl 4,4,4,trifluoromethyl 3-(trichloromethyl)crotonate,4,4,4,trifluoromethyl 3-(trichloromethyl)crotonic acid, Ethyl 4,4,4trichloromethyl 3-(trifluoromethyl)crotonate, 4,4,4 trichloromethyl3-(trifluoromethyl)crotonic acid Ethyl 4,4,4 trichloromethyl3-(trichloromethyl)crotonate, 4,4,4 trichloromethyl3-(trichloromethyl)crotonic acid, 4,4,4, tribromomethyl3-(trifluoromethyl)crotonic acid, Ethyl 4,4,4 tribromomethyl3-(trifluoromethyl)crotonate, 4,4,4 trifluoromethyl3-(tribromomethyl)crotonic acid, 4,4,4 trichloromethyl3-(tribromomethyl)crotonic acid, 4,4,4, triiodomethyl3-(trifluoromethyl)crotonic acid, Ethyl 4,4,4 triiodomethyl3-(trichloromethyl)crotonate, 4,4,4 trifluoromethyl3-(triiodomethyl)crotonic acid, 4,4,4 trichloromethyl3-(triiodomethyl)crotonic acid.

The effect of the crotonate compounds varied with their concentration.In accordance with this invention the crotonate compounds will beeffective in fermentation process with a concentration of the purecompound of as little as 50 ppm to inhibit the growth of the butryigens.Effective concentrations may be reduced significantly with the use ofdelivery systems and agents that improve the uptake of the compounds bythe microorganisms. In most cases concentrations in excess of 1000 ppmare avoided so as to not hinder the growth of the microorganisms thatare producing the light oxygenates such as ethanol.

A broad aspect of this invention is a method of restricting theproduction of butyrate and butanol in an anaerobic fermentation of a gassubstrate that comprises at least one of CO and/or a mixture of CO₂ withhydrogen. The method passes the gas stream to an anaerobic fermentationzone containing at least one species of anaerobic microorganism capableof producing an oxygenated liquid product other than or in addition tobutyrate and butanol. At least a portion of the gas stream is convertedto the liquid product by contact of the microorganism in thefermentation zone with the gas stream. A di-halogen substitutedcrotonate ester and/or acid is added to the fermentation liquid as abutyl retardant in an amount effective to restrict production ofbutyrate and butanol. The method withdraws a fermentation liquidcontaining the liquid product from the fermentation zone and recoversthe liquid product from the fermentation liquid. The fermentation zonewill usually contain multiple species of microorganisms, typically ahomoacetogenic microorganism for the production of a liquid product anda butyrigenic microorganism that produces butyrate and/or butanol. Thefermentation zone may also contain heteroacetogenic microorganisms thatproduce a butyrate or butanol as well as a liquid product such as aceticacid and/or ethanol.

Another broad aspect of this invention is a method of producing ethanolby the fermentation of a gas stream that contains CO and/or a mixture ofCO₂ with hydrogen using a homoacetogenic microorganism to convert thegas stream wherein the production of butyrate is inhibited. The methodpasses the gas stream to a fermentation zone containing a homoacetogenicmicroorganism and a fermentation liquid. Contact of the homoacetogenicmicroorganism with the gas stream produces ethanol in the fermentationzone. A di-halogen substituted crotonate acid or ester is introducedinto the fermentation liquid in a sufficient amount to inhibit thegrowth of butyrigens and the production of butyrate and butanol. Thebutyl retardant concentration may range between 10 and 1000 ppm on anintermittent or continual basis. The method withdraws ethanol containingfermentation liquid from the fermentation zone and an ethanol product isrecovered from the fermentation liquid.

In another form this invention is a method for producing ethanol by thefermentation of a gas stream that contains CO and/or a mixture of CO₂with hydrogen using a homoacetogenic microorganism to convert the gasstream to ethanol. The method passes the gas stream to a fermentationzone containing the homoacetogenic microorganism and a fermentationliquid that converts the gas stream to ethanol by contact with thehomoacetogenic microorganism. A butyl retardant comprising a di-fluorosubstituted crotonate compound is added to the fermentation zone todisrupt the growth of butyrate producing microorganisms. The butylretardant is added in an amount that produces a concentration of thedi-fluoro substituted crotonate compound in a range of between 50 and1000 ppm. The method withdraws the ethanol containing fermentationliquid from the fermentation zone and an ethanol product is recoveredfrom the fermentation liquid. In a preferred form of the invention thebutyl retardant comprises Ethyl 4,4,4, Triflouromethyl3(trifluromethyl)crotonate and it is added to the fermentation zone at aconcentration of 50 to 500 ppm.

FIGURES

FIG. 1 is diagram illustrating some key enzymes products andintermediate products in butyrigen metabolism for producing butanol andbutyrate.

FIG. 2 is a bar graph showing the product distribution fromfermentations with heteroacetogenic bacteria in the presence of varyingconcentrations of a crotonate derivative.

FIG. 3 is a plot showing the concentration of acetate and butyratecompounds over time in a continuous fermenter run along with the opticaldensity of the fermentation liquid.

FIG. 4 is a plot showing the production and production rate of butyratecompounds over a selected time period from the continuous fermenter runof FIG. 3.

FIG. 5 is an expanded portion of the plot of FIG. 3 betweenapproximately 500 hours and 640 hours.

FIG. 6 is an expanded portion of the plot of FIG. 3 betweenapproximately 1660 hours and 1900 hours.

DETAILED DESCRIPTION Definitions

Butyrigens refer to microorganisms that under anaerobic conditionsproduce compounds having four carbon atoms such as butyrates and butanoland the term can also include longer chain (C₆-C₈) organic acids andalcohols.

Butyl retardant refers to a compound that is used to inhibit or kill thebutyrigens. The term contemplates the activity of the compound as abactericide or bacteriostatic agent.

Butyl impurity refers to any molecule that has a total of four or morecarbon atoms in its structure with the carbon atoms arranged as a chain.

Light oxygenates refers to any molecule that has two or three carbonatoms and at least one carbon-oxygen bond.

General Description

This invention applies to anaerobic fermentations to produce lightoxygenates such as ethanol, acetic acid, propanol, and propionic acidusing a gas substrate comprising carbon monoxide and hydrogen, and thegas will typically contain carbon dioxide and nitrogen. Syngas is onesource of such a gas substrate. Syngas can be made from manycarbonaceous feedstocks. These include sources of hydrocarbons such asnatural gas, biogas, gas generated by reforming hydrocarbon-containingmaterials, peat, petroleum coke, and coal. Other sources for productionof syngas include waste material such as debris from construction anddemolition, municipal solid waste, and landfill gas. Syngas is typicallyproduced by a gasifier. Any of the aforementioned biomass sources aresuitable for producing syngas. The syngas produced thereby willtypically contain from 10 to 60 mole % CO, from 10 to 25 mole % CO₂ andfrom 10 to 60 mole % H₂. The syngas may also contain N₂ and CH₄ as wellas trace components such as H₂S and COS, NH₃ and HCN. Other sources ofthe gas substrate include gases generated by petroleum and petrochemicalprocessing. These gases may have substantially different compositionsthan typical syngas, and may be essentially pure hydrogen or essentiallypure carbon monoxide. Also, the substrate gas may be treated to removeor alter the composition including, but not limited to, removingcomponents by sorption, membrane separation, and selective reaction.Components may be added to the gas substrate such as nitrogen oradjuvant gases such as ammonia and hydrogen sulfide. The term syngaswill be used herein and will be intended to include these other gassubstrates.

This invention will use homoacetogenic microorganisms and fermentationconditions particularly selected for the production of light oxygenatesand preferably selected for the production of ethanol. Bioconversions ofCO and H₂/CO₂ to acetic acid and ethanol and other products are wellknown. Suitable microorganisms live and grow under anaerobic conditions,meaning that gaseous and dissolved oxygen is essentially absent from thefermentation zone. A concise description of biochemical pathways andenergetics for acetogenic bioconversions have been summarized by Das, A.and L. G. Ljungdahl, Electron Transport System in Acetogens and byDrake, H. L. and K. Kusel, Diverse Physiologic Potential of Acetogens,appearing respectively as Chapters 14 and 13 of Biochemistry andPhysiology of Anaerobic Bacteria, L. G. Ljungdahl eds,. Springer (2003).Any microorganisms that have the ability to produce ethanol byconverting the syngas components: CO, H₂, CO₂ individually or incombination with each other or with other components that are typicallypresent in syngas may be utilized. Suitable microorganisms and/or growthconditions may include those disclosed in U.S. Pat. No. 7,704,723entitled “Isolation and Characterization of Novel Clostridial Species,”which discloses a biologically pure culture of the microorganismClostridium ragsdalei having all of the identifying characteristics ofATCC No. BAA-622 which is incorporated herein by reference in itsentirety. Clostridium ragsdalei may be used, for example, to fermentsyngas to ethanol.

Suitable microorganisms include: Clostridium Ljungdahlii, with strainshaving the identifying characteristics of ATCC 49587 (U.S. Pat. No.5,173,429) and ATCC 55988 and 55989 (U.S. Pat. No. 6,136,577) that willenable the production of ethanol as well as acetic acid; Clostridiumautoethanogemum sp. nov., an anaerobic bacterium that produces ethanolfrom carbon monoxide. Jamal Abrini, Henry Naveau, Edomond-Jacques Nyns,Arch Microbiol., 1994, 345-351; Archives of Microbiology 1994, 161:345-351; and Clostridium Coskatii having the identifying characteristicsof ATCC No. PTA-10522 filed as U.S. Ser. No 12/272,320 on Mar. 19, 2010.All of these references are incorporated by reference herein in theirentirety.

The invention can provide benefit for any type of fermentation zone.Suitable fermentation zones are typically referred to as a bioreactor.The term “bioreactor” includes a fermentation device consisting of oneor more vessels and/or towers or piping arrangements, which includes theContinuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR),Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, MembraneReactor such as Hollow Fiber Membrane Bioreactor (HFMBR), Static Mixer,or other vessel or other device suitable for gas-liquid contact.

Typical bioreactors have the arrangement of a suspended cell typebioreactor or membrane supported bioreactor. In a suspended cell typebioreactor the fermentation liquid contains the microorganisms insuspension as the gas substrate passes through the fermentation liquidto effect contacting between the gas and the microorganisms byabsorption of the gas into the liquid and uptake of the dissolved gas bythe microorganism. Suspended cell bioreactors typically take the form ofa continuous stirred tank where impellers provide mechanically mix thegas substrate and the fermentation liquid or a bubble column bioreactorwhere the injection of the substrate into the gas promotes mixing of thegas and liquid.

A membrane supported bioreactor utilizes a solid surface upon which togrow the microorganisms as a biofilm or a concentration of cells thatthe substrate gas contacts. One membrane bioreactor, as shown in US20080305539 A1, grows the biofilm on one side of the membrane and indirect contact with the fermentation liquid while the substrate gaspermeates into contact with the biofilm from the opposite side of themembrane. US 20090215163 A1 discloses the opposite arrangement for amembrane supported bioreactor where one side of the membrane retains themicroorganisms in direct contact with the gas substrate while thefermentation liquid permeates from the opposite of the membrane and intocontact with the microorganisms. Either type of membrane supportedbioreactor is suitable for use with this invention.

When using the invention with a suspended cell bioreactor thefermentation liquid will include a suspension of microorganisms andvarious media supplements. The various adjuvants to the fermentationliquid may comprise buffering agents, trace metals, vitamins, salts etc.Adjustments in the fermentation liquid may induce different conditionsat different times such as growth and non-growth conditions which willaffect the productivity of the microorganisms. Previously referencedU.S. Pat. No. 7,704,723, the contents of which are hereby incorporatedby reference, discloses the conditions and contents of suitablefermentation menstruum for bioconversion of CO and H₂/CO₂ usinganaerobic microorganisms. Other methods, operating conditions and mediafor operating bioreactors to produce ethanol are described in theliterature that includes those described in WO2007/117157,WO2008/115080, U.S. Pat. No. 6,340,581, U.S. Pat. No. 6,136,577, U.S.Pat. No. 5,593,886, U.S. Pat. No. 5,807,722 and U.S. Pat. No. 5,821,111,each of which is incorporated herein by reference.

The fermentation is carried out under appropriate conditions thatinclude pressure, temperature, gas flow rate, liquid flow rate, mediapH, media redox potential, agitation rate (if using a continuous stirredtank reactor), inoculum level, and maximum gas substrate concentrationsto ensure that CO in the liquid phase does not become limiting, andmaximum product concentrations to avoid product inhibition. Suitableconditions are described in WO02/08438, WO07/117,157 and WO08/115,080.Typically, the fermentation liquid and the microorganisms in thefermentation zone include a suitable temperature in the range of between25° C. and 60° C., and more frequently in the range of about 30° C. to40° C. Other conditions of fermentation include the density ofmicroorganisms, fermentation liquid composition, and liquid depth, whichare all preferably sufficient to achieve the sought conversion ofhydrogen and carbon monoxide.

Fresh liquid media containing nutrients will typically enter thebioreactor on a continual basis. The addition of the fresh media and thewithdrawal of fermentation liquid provide a continual withdrawal of cellmass from the bioreactor. As the fermentation continues to generate cellmass, the removal and addition rate of media and fermentation liquidwill establish a mean cell retention time in the bioreactor. Mean cellretention times for most fermentation zone are typically in a range offrom 2 to 7 days.

The butyl retardant as described in this invention is a class ofcompounds that has the effect of inhibiting or stopping the productionof acids or alcohols with four or longer chain carbon atoms bymicroorganisms in a fermentation zone while not substantiallyinterfering with the conversion of syngas to lower carbon numberoxygenates such C2 and C3 acids and alcohols. Useful butyl retardantsare any compounds that can interfere with the butyl impurity productionof one or more butyrigens. Known butryrigens include the strictbutyrigens that produce essentially only butyl impurities andheteroacetogens that can produce ethanol and acetate along with butylimpurities.

Butryrigens are characterized by having a critical step of in theirmetabolic pathway for the conversion of crotonyl CoA to butyryl CoA.FIG. 1 shows generalized pathways for butyrigen metabolism and that thepathways for production of butanol and butyrate differ only in thestarting substrates. The starting substrates to the production ofbutanol and butyrate include ethanol and acetic acid through path 1,sugar through path 2 and 3, and syngas through path 4.

Butyrigens such as C. acetobutylicum, make a butyrate as a primaryproduct or as a smear of short-chain fatty acid products as part oftheir primary metabolism in which several acids and their correspondingsolvents are also produced (FIG. 1, Path 2). These organisms use sugarsor proteins as substrates for metabolite production. Other butyrigens,such as various Eubacterium strains and Roseburia are strictlybutyrogenic and produce large amounts of the acid by using a differentenzyme to generate the final product, butyrate (FIG. 1, Path 3). Theseclasses of butyrigens are predicted to be less problematic than thosethat use Path 1 and Path 4 since they may be metabolically disadvantagedunder the growth conditions predicted to exist in large-scale syngasreactors.

Path 1 (FIG. 1) encompasses C. kluyveri and similar metabolic types oforganisms that can use ethanol, acetate and hydrogen to make C₄ andhigher chained acids. C. kluyveri can reside in a reactor and form acommensal relationship with other organisms, thus potentially making itmetabolically advantaged and which can lead to persistent infections ifthe fermenter's environmental conditions are not altered. Thus, of thenon-syngas using butyrigens, C. kluyveri and similar types pose a highrisk for long-term, persistent contamination since they can readily makeC₄ or higher acids from ethanol and acetate or two acetates. Thisreaction is also thermodynamically advantaged under certain conditions.

Finally, organisms that use Path 4 (FIG. 1) are those that can grow onboth sugars and syngas and produce a variety of short chain and higherchain acids and alcohols. These are broadly classified as theheteroacetogens and members include C. carboxidivorans and C. drakei.These heteroacetogens can use either sugars as well as syngas substratesand can also make C₄ acids and solvents from C2 subunits. Theheteroacetogens pose a slightly higher risk than other butyrigens, sincethey may be metabolically advantaged under the low redox, high dissolvedsyngas conditions that would exist in a large-scale fermentation zone.

As with the strict butyrigens, the heteroacetogens use a common pathwayto generate C₄ compounds. The entry point into butyrigenesis, which is ahighly conserved pathway except for a few endpoint reactions among allbutyrigens, occurs when two acetyl-CoAs condense to form acetoacetyl-CoAby a reaction with thiolase. This is followed by a hydroxylation at oneof the carbonyls to form S3-hydroxybutyryl-CoA (FIG. 1).Hydroxybutyryl-CoA is dehydrated by crotonase to form crotonyl-CoA. Theprevious reaction to form 3-HBCoA can be achieved by a variety ofdehydrogenases and does not generate any energy for the cell, but itdoes regenerate some oxidized cofactor. Once the crotonyl-CoA isgenerated the cell becomes susceptible to inhibitors since most, if notall, of the energy derived from butyrogenesis comes from the conversionof crotonyl-CoA to butyryl-CoA. All of these pathways are stopped bydisrupting the critical step of converting the crotonyl CoA to butyrylCoA.

It was found that a class of crotonate compounds would act as butylretardants in the production of light oxygenates from syngas. It isbelieved that these compounds interrupt this critical step in thebutyrigen metabolism. Specifically crotonate esters and acids having adisubstitution of halogenenated methyl groups were found to inhibit thegrowth of strict butyrigens and heteroacetogens. At the same time, thiscrotonate ester did not interfere with the production of lightoxygenates such as ethanol by a homoacetogen. Importantly, thedeleterious effect on the butyrigen occurred in vivo thereby making thisretardant suitable for direct use in fermentations.

Although not wishing to be bound by any theory, the halogenated moietiesof the molecule are believed to disrupt the enzyme activity at theessential Crotonyl CoA to Butyryl CoA step. This disruption is believedto be caused by the dihalogenated crotonate compounds ability to mimicthe crotonate molecule and interfere with the catalytic activity of theenzyme. On this basis, the di-halogenated crotonate esters and acidswill generally have the effect of interrupting this step.

These butyrigen retardants operate at a relatively low concentrationlevel. At concentrations as low as 50 ppm in a continuous fermentationthe crotonate derivatives had beneficial effects on reducing butanol andbutyric acid while not disrupting the production of acetic acid andethanol. In fact it has been discovered that crotonate derivatives donot begin to impact homoacetogen growth until reaching concentrations of1000 ppm in the fermentation liquid. Thus, the amount of the crotonatederivative added to the fermentation zone can be adjusted to maintain itin a range that will effectively retard the growth of the butyrigenswithout inhibiting the growth of desired homoacetogens. As a result useof the butyl retardant can be tuned to curtail the production of butylimpurities while not harming the production of ethanol, acetic acid andother light oxygenate products.

The butyl retardant may be added to a fermentation zone in a variety ofways. It may be injected in a desired dosage directly into thefermentation zone. The butyl retardant may also be mixed with the freshmedia input or a recycle stream from the fermentation zone to promotebetter mixing of the butyl retardant in the fermentation zone.

These butyl retardants and readily miscible in simple hydrocarbons andother non-aqueous solvents. For example common aromatic hydrocarbonssuch as xylenes, toluene etc. or aliphatic hydrocarbons such as hexanedissolve these butyl retardants. Hence these can be dissolved in suchhydrocarbon and other non-aqueous solvents and made into microemulsionsby well-known techniques by addition of suitable surfactants andcosolvents. These microemulsion particles are typically 0.1 micron indiameter and can be readily dispersed into the fermentation medium.There these emulsion particles will contact with the butyrigens anddeliver the butyl retardants to the organisms. The major advantage ofsuch microemulsion delivery system is that they are effective in muchlower dosages because they can protect the active compounds such as thebutyl retardants and also deliver them to the cell surface which is theprimary target. Methods for making such microemulsions and their usagein general biocide formulations have been described in U.S. Pat. No.6,096,225 by Yang et al. In a suspended cell or planktonic typefermentation the butyl retardant will be effective when introduced as asingle dose at a concentration of from 10 to 1000 ppm with concentrationlevels of 50 to 500 ppm being preferred for most applications. Theeffective concentrations will be influenced by the delivery system withmicroemulsion systems having effective concentration below 50 ppm anddown to 10 ppm or lower. Preferably these dosages are calculated basedon the mean cell retention time of the bioreactor such that the dosagewill have a duration of at least 2 days.

It is possible to add the butyl retardants in varied amounts in responseto monitoring of butyl impurity production and the C₂ product output andmaking adjustments in addition and concentration depending upon theproduction of the butyl impurities. In this respect the desired amountof butyl retardant can be added to maintain a desired concentration in afermentation zone or in response to monitoring the presence of butyrigencontamination. Thus, the butyl retardant may be used continually orintermittently to prevent or reverse the effect of butyrigen growth. Ifdesired, the butyl retardant may be added at the start of the of afermentation process. In this manner the butyl retardant acts as aprophylactic measure to prevent butytrigen contamination from takinghold in the fermenter. The addition may be continued throughout thefermentation process by continuous or intermittent injection of thebutyl retardant into the fermentation zone. In such cases a relativelylow butyl retardant dosage can be effective. In particular, intermittentdosages at a concentration level of 50 ppm or preferably 50 to 100 ppmon a frequency of 5 to 10 days may be used. In the absence of recoveryand recirculation of liquid containing the butyl retardant, the butylretardant will wash out of the bioreactor at a rate determined by themean cell retention time.

Regardless of the delivery system any method may be used to determinethe presence of butyrigens and the effectiveness of the butyl retardant.Monitoring of the product output for the presence of butyl impuritiesfrom the fermentation zone can provide an indication of butyrigencontamination. Preferably the fermentation liquid will undergo periodicsampling for detection of butyl impurities.

Most often the butyl retardant is added in response to the detection ofthe butyrigens. In this case the butyl retardant is added in sufficientamount to produce a single dose in a concentration of 100 to 1000 ppm inthe fermentation zone, with a dose in the range of 500 to 1000 ppm beingpreferred. A desired concentration of crotonate compound may bemaintained until the presence of the butyrigens has been reduced to adesired level as typically indicated by the production of butylimpurities from the fermentation zone. Once sufficient butyrigens havebeen reduced to a level that produces an acceptable fermentation zoneproduct, the crotonate compound can be allowed to wash out of thefermentation zone.

The butyl retardant can be introduced to achieve a desired reduction inthe amount of butyl impurities. Ideally, the butyl impurities in the inthe fermentation liquid are reduced to zero, however, the fermentationliquid will typically contain some amount of butyl contamination. Inmost cases the butyl retardant will be used as necessary to keep thebutyrate and butanol concentration in the ethanol containingfermentation liquid below 0.1% and preferably below 0.01%.

EXAMPLES Examples 1-5

A variety of crotonate derivatives were tested for their bactericide orbacteriostatic activity. The tested compounds are identified in Table 1.

TABLE 1 A. Ethyl 4,4,4, trifluoromethyl 3-(trifluoromethyl) 236.12crotonate B. Ethyl-3-methylamino-4,4,4-trifluorocrotonate 182.14 C.Tert-butyl crotonate 142.20 D. 4,4,4-Trifluoro-3-(trifluoromethyl)crotonic acid 208.06 E. Ethyl 4,4,4-trifluorocrotonate 168.12 F.Ethyl-2-methyl-4,4,4-trifluorocrotonate 197.16 G.4,4,4-trifluorocrotonic acid 140.06 H. Ethyl 3-aminocrotonate 129.16

Example 1

These compounds were first tested to determine their effect on a knownhomoacetogen. Each compound was tested in a series of batch experimentsto determine the growth response of a homoacetogen to the presence ofthe compound at varying concentrations. The batch experiments were allconducted by anoxically filling a Balch tube with 5 ml of a fermentationmedium having the composition given in Tables 2 and 3. To expediteresults, these batch experiments used fructose as the growth nutrientsource for bacteria. Thus, the media included a 5 g/L of fructose.

TABLE 2 Fermentation Medium Compositions Components Amount per literMineral solution, See Table 3(a) 25 ml Trace metal solution, See Table3(b) 10 ml Vitamins solution, See Table 3(c) 10 ml Yeast Extract 0.5 gAdjust pH with NaOH 6.1 Reducing agent, See Table 3(d) 2.5 ml

TABLE 3(a) Mineral Solution Components Concentration (g/L) NaCl 80 NH₄Cl100 KCl 10 KH₂PO₄ 10 MgSO₄•7H₂O 20 CaCl₂•2H₂O 4

TABLE 3(b) Trace Metals Solution Components Concentration (g/L)Nitrilotriacetic acid 2.0 Adjust the pH to 6.0 with KOH MnSO₄•H₂O 1.0Fe(NH₄)₂(SO₄)₂•6H₂O 0.8 CoCl₂•6H₂O 0.2 ZnSO₄•7H₂O 1.0 NiCl₂•6H₂O 0.2Na₂MoO₄•2H₂O 0.02 Na₂SeO₄ 0.1 Na₂WO₄ 0.2

TABLE 3(c) Vitamin Solution Components Concentration (mg/L)Pyridoxine•HCl 10 Thiamine•HCl 5 Roboflavin 5 Calcium Pantothenate 5Thioctic acid 5 p-Aminobenzoic acid 5 Nicotinic acid 5 Vitamin B12 5Mercaptoethanesulfonic acid 5 Biotin 2 Folic acid 2

TABLE 3(d) Reducing Agent Components Concentration (g/L) Cysteine (freebase) 40 Na₂S•9H₂O 40

Each tube was inoculated with 0.5 ml of the same strain of C.autoethanogenum bacteria seed culture inoculum. The tubes weremaintained at a temperature of 37° C. Twenty one hours after theinoculation of the tube with the bacteria, the different crotonatederivatives from Table 1 were added to different tubes in the amountsindicated in Table 4 This was at a time of early to mid-log phase growthfor the bacteria. Each fermentation of the bacteria in the media at thedifferent concentration of crotonate derivatives were allowed toprogress and were monitored to determine the bacteria growth at selectedintervals of time varying from approximately 20 hours to 190 hours.Growth at the intervals was measured by reading the optical density (OD)of the fermentation liquid. Optical density was measured using aSpectronic Spec 20 (Thermo Spectronic at a wavelength of 600 nm. The ODof the Balch tube culture was measured directly in the tube usingabsorbance mode on the Spec 20 machine. The machine was set to zeroabsorbance by first measuring and adjusting the setting to zeroabsorbance using media only as a blank. This process was repeated ateach indicated time point throughout the experiment. The ability of atube fermentation at a particular concentration of crotonate derivativeto reach a predetermined optical density was recorded in Table 4. Table4 indicates the presence of bacterial growth to an OD of approximately 2or above with a “+”, and to an optical density in a range of 0.5 to 1.5as a “+/−”, and indicates the failure of bacterial growth to reach anoptical density of approximately 0.5 as a “−”.

TABLE 4 ppm 0 1 5 10 50 100 500 1000 5000 10000 A + + + + + + + + − −B + + + + + + + + +/− − C + + + + + + − D + + + + +/− − − E + + + + + −− F + + + + + +/− − G + + + + +/− − − H + + + + + + +

As the Table 4 indicates, the homoacetogenic bacteria tolerated thepresence of the tested crotonyl compounds up to concentrations of about5000 ppm.

Example 2

Compounds A and D were again tested to determine their effect on anotherknown homoacetogen. Each compound was tested in a series of batchexperiments to determine the growth response of the homoacetogen to thepresence of the compound at varying concentrations. These batchexperiments were again all conducted by anoxically filling a Balch tubewith 5 ml of a fermentation medium having the composition given inTables 2 and 3. To expedite results, these batch experiments usedfructose as the growth nutrient source for bacteria. Thus, the mediaincluded 5 g/L of fructose.

Each tube was inoculated with 0.5 ml of the same strain of C. coskatiibacteria seed culture inoculum. The tubes were maintained at atemperature of 37° C. Twenty one hours after the inoculation of the tubewith the bacteria, the different crotonate derivatives from Table 1 wereadded to different tubes in the amounts indicated in Table 5 This was ata time of early to mid-log phase growth for the bacteria. Eachfermentation of the bacteria in the media at the different concentrationof crotonate derivatives were allowed to progress and were monitored todetermine the bacteria growth at selected intervals of time varying fromapproximately 20 hours to 50 hours. Growth at the intervals wasdetermined by measurement of the OD in the manner described inExample 1. The ability of a tube fermentation at a particularconcentration of crotonate derivative to reach a predetermined opticaldensity was recorded in Table 5. Table 5 indicates the presence ofbacterial growth to an OD of approximately 1.6 or above with a “+”. Allthe samples of C. Coskatii reached a relatively high OD after a time ofonly 50 hours.

TABLE 5 ppm 0 100 500 1000 A + + + + D + + + +

Example 3

Similar experiments were conducted to make a preliminary determinationas to which of the crotonate derivatives would act as butyrigenretardants. For this purpose the selected compounds were tested again ina series of batch experiments to determine the growth response ofbutyrigens to the presence of the compound at varying concentrations.The batch experiments were all conducted by anoxically filling a 20 mlBalch tube with 5 ml of a fermentation medium having the compositiongiven in Tables 2 and 3. The media included 0.5 v/v final concentrationof a fructose substrate. For each of the crotonate concentrationstested, duplicate test tubes were prepared.

Each tube was inoculated with 0.5 ml seed culture inoculum of the strictbutyrigen Clostridium tyrobutyricum. The tubes were maintained at atemperature of 37° C. Twenty one hours after the inoculation of the tubewith the bacteria, the different crotonate derivatives from Table 1 wereadded to different tubes in the amounts indicated in Table 6. This wasat a time of early to mid-log phase growth for the bacteria. Eachfermentation of the bacteria in the media at the different concentrationof crotonate derivatives were allowed to progress and were monitored todetermine the bacteria growth at selected intervals of time varying fromapproximately 20 hours to 54 hours. Growth at the intervals was measuredby reading the optical density (OD) of the fermentation liquid. Opticaldensity was measured as described in Example 1. The ability of a tubefermentation at a particular concentration of crotonate derivative toreach a predetermined optical density was recorded in Table 6. Table 6indicates the presence of bacterial growth to an OD of approximately 1.8or above with a “+”, and to an optical density in a range of 0.7 to 1.5as a +/−, and indicates the failure of bacterial growth to reach anoptical density of approximately 0.7 as a “−”.

TABLE 6 ppm 0 100 500 1000 A + + +/− − B + + + + C + + + + D + + + +E + + + + F + + + + G + + + +

Example 4

Similar experiments were conducted to determine the effect of thecrotonate derivatives on heteroacetogenic bacteria. Selected crotonatederivatives were tested in a series of batch experiments to determinethe growth response of butyrigens to the presence of the compounds atvarying concentrations. The batch experiments were all conducted byanoxically filling a 20 ml Balch tube with 5 ml of a fermentation mediumhaving the composition given in Tables 2 and 3. The media included 0.5v/v final concentration of a fructose substrate. For each of thecrotonate concentrations tested, duplicate test tubes were prepared.

Each tube was inoculated with 0.5 ml of the heteroacetogenic bacteriaClostridium Carboxydivorans. The tubes were maintained at a temperatureof 37° C. At a time 20 hours after the inoculation of the bacteriadifferent crotonate derivatives from Table 1 were added to differenttubes in the amounts indicated in Table 7. This was at a time of earlyto mid-log phase growth. Each fermentation of the bacteria in the mediaat the different concentration of crotonate derivatives were allowed toprogress and were monitored to determine the bacteria growth at selectedintervals of time varying from approximately 20 hours to 54 hours.Growth at the different intervals was measured by reading the opticaldensity (OD) of the fermentation liquid. Optical density was measured asdescribed in Example 1. The ability of a tube fermentation at aparticular concentration of crotonate derivative to reach apredetermined optical density was recorded in Table 7. Table 7 indicatesthe presence of bacterial growth to an OD of approximately 1.8 with a“+”. For these experiments the +/− indicates that one duplicate testtube showed a + indication and the other showed an optical density ofless than 0.5.

TABLE 7 ppm 0 100 500 1000 A + + + + D + + + +/−

Product profiles were determined on the C. carboxidivorans cultures thatgrew in the presence of compound A. A GC-MSD was performed on 54 hourcultures and product distributions were calculated based on theresultant areas of the output (See FIG. 2). In cultures with no compoundA addition, the distribution of C₂, C₄ and C₆ acids and alcohols wassimilar in both samples. As the compound A concentration increased theamount of C₂, particularly the alcohols, increased. At 500 and 1000 ppmvirtually all the product observed was the C₂ alcohol. Thus the presenceof compound A shows the inhibitory effect on C₄ and longer chain alcoholand acid production. Thus, such crotonate derivatives can effectivelylimit the amount of butyl impurity accumulation in the fermentation zonefrom heteroacetogens which can compete effectively in syngas cultureswith homoacetogenic bacteria.

Example 5

Compound A was tested again in set of experiments in all respects thesame as that described for Example 4 except that compound A was added toBalch tubes in the amounts indicated in Table 7 at a time 5.5 hoursafter the inoculation of the bacteria. This was at a time in early logphase growth. In this case the Balch tube that contained no compound Areached an OD of 2 after 29 hours or less. After about 54 hours the tubefermentations that received 100 ppm of compound A had one tube reach anOD of at least 2 while the duplicate tube failed to reach an OD of 1.For all the fermentation tubes that received 500 and 1000 ppm ofcompound A, the OD failed to rise above about 0.5.

Examples 1-5 showed that compounds A and D were both tolerated by thehomoacetogens at concentration of up to 1000 ppm. Both of compounds Aand D showed inhibition effect for reducing butyrigen growth. Thus, thedihalogenated crotonyl esters and acids were shown to have usefulin-vivo inhibition effects for reducing butyl impurities inhomoacetogenic fermentations for the production of ethanol and aceticacid.

Examples 6-10

Additional experiments were conducted to determine the effect of thecrotonyl derivatives in continuous fermentations containing butyrigencontamination. Compounds A and D were tested in a 2-L fermentercontaining a seed culture from a 10,000 gallon bioreactor used in alarge scale fermentation run. The large scale fermentation run had afermentation liquid volume of approximately 8,000 gallons. The largescale fermentation run was grown from an inoculation of ClostridiumAutoethanogenum that showed a significant presence of buytrigen by thepresence of 0.5% butyl compounds in its products.

To initiate the 2 liter fermentation a seed culture from the large scalefermenter was introduced into the 2 liter fermenter and grown on yeastextract to an OD greater than 1 at which time a substantial productionof butyrigens was observed. The 2 liter fermenter was a SartoriusBiostat B Series fermenter that operated as a continuously stirred tankwith a mixing speed of 200 rpm. After 24 hours of initial growth a gasstream having a composition 38% CO, 38% H2, 15% CO₂ and the balance CH₄was introduced into the fermenter. The fermentation was conducted at atemperature of 37° C., a pH of 5.30±0.05 and contained approximately 2liters of fermentation media having a composition found in Table 8.Fresh fermentation media was continually added to the 2 liter fermenterat rate sufficient to establish a mean cell retention time of 5.8 days.The fermentation was allowed to progress and different concentrations ofthe compounds A and D were added to the fermentation as intermittentinjections at different times. Each injection was allowed to wash out ofthe fermenter before next injection. The presence of acetate andbutyrate compounds in the fermenter along with the OD of thefermentation media are shown in FIG. 3.

TABLE 8 2-liter Fermentation Medium Composition Components Amount perliter Mineral solution, See Table 7(a) 25 ml Trace metal solution, SeeTable 7(b) 10 ml Vitamins solution, See Table 7(c) 10 ml Yeast Extract 0g Adjust pH with NaOH 6.1 Reducing agent, See Table 2(d) 2.5 ml

TABLE 7(a) Mineral Solution Components Concentration (g/L) NaCl 0 NH₄Cl100 KCl 10 KH₂PO₄ 20 MgSO₄•7H₂O 5 CaCl₂•2H₂O 2

TABLE 7(b) Trace Metals Solution Components Concentration (g/L)Nitrilotriacetic acid 0 pH 2.0 with 12.1 N HCL MnSO₄•H₂O 0.377Fe(NH₄)₂(SO₄)₂•6H₂O 0 CoCl₂•6H₂O 0.358 ZnSO₄•7H₂O 1.96 NiC1₂•6H₂O 0.078Na₂MoO₄•2H₂O 0 Na₂SeO₄ 0.1 Na₂WO₄ 0.118 Fe(SO4)•7H2O 3.657 pH 2.0 with12.1 N HCL

TABLE 7(c) Vitamin Solution Components Concentration (mg/L)Pyridoxine•HCl 0 Thiamine•HCl 10 Riboflavin 0 Calcium Pantothenate 10Thioctic acid 0 p-Aminobenzoic acid 0 Nicotinic acid 0.5 Vitamin B12 0Mercaptoethanesulfonic acid 0 Biotin 5 Folic acid 0

TABLE 7(d) Reducing Agent Components Concentration (g/L) Cysteine (freebase) 40 Na₂S•9H₂O 0 Clerol antifoam 0.02

Example 6

After approximately 500 hours of fermenter operation compound A wasadded directly into the fermenter to a concentration of 500 ppm when theOD was 1.82 and butyrate concentration was 4.5 g/L. Followingintroduction of the compound, the OD decreased rapidly by almost 50% inthe first 24 hours and the butyrate concentration decreased by 20% inthe same time period. Also in the first 24 hours the butanol contentincreased rapidly by 6-fold to 0.68 g/L, indicating rapid conversion ofthe butyric acid to butanol. The rate of conversion of the butyric acidand the dilution rate of the media combined to dramatically decrease thebutyrate production rate until it was brought down to zero within 80hours of initially adding compound A. (See FIG. 4) In addition the ODcontinued to decrease to a low of 0.54 after 80 hours. Approximately 140hours after the 500 ppm addition of compound A the OD nearly recoveredto its original level with an increase in ethanol of over 100% and adramatic rise in acetic acid production. (See FIG. 5) The concentrationof the C₂ and C₄ alcohols and acids are shown in Table 9.

TABLE 9 Addition OD Ethanol Acetic acid n-Butanol Butyric acid Hr @ 600nm g/L g/L g/L g/L 0.0 1.82 0.026 3.237 0.109 4.503 24.0 1.09 0.2662.728 0.681 3.600 49.0 0.76 0.255 2.333 0.601 3.074 80.0 0.54 0.2121.959 0.497 2.542 121.0 0.75 0.345 2.446 0.513 1.842 145.0 1.17 0.4414.082 0.311 1.645

Example 7

After approximately 700 hours of fermenter operation and the washout ofthe first 500 ppm addition of compound A, another 50 ppm of compound Awas added directly into the fermenter. A decrease in OD and butyrateconcentration (See FIG. 3) followed the 50 ppm addition and continuedfor at least for the first 3 days. At the same time there was acontinued increase in the concentration of ethanol and acetic acid.

Example 8

After approximately another 8 days and the washout of the 50 ppmaddition of compound A, the butyrate concentration increased to 2 g/L.At this time another 100 ppm of compound A was added directly into thefermenter approximately 890 hours into the run. Upon addition ofcompound A the ethanol concentration began to immediately increase.After about 24 hours, the butyrate concentration began to decrease againdeclining almost 50% over the next 4 days. (See FIG. 3)

The results of Examples 5-8 show that in a 2-L butyrogenic reactorcompound A had an inhibitory and bactericidal effect on the butyrigenpopulation shortly after addition. In all three trials, addition ofcompound A showed a decrease in the first 24 hours in both OD andbutyrate production with an increase in C2 production. The addition ofthe butyrigen retardant was shown to have beneficial effects atconcentrations as low as 50 ppm.

Example 9

Fermenter operation was continued under the same operating conditionsfollowing the 100 ppm addition of compound A. After allowing 20 days forthe complete wash out of compound A, 100 ppm of compound D was addeddirectly into the fermenter approximately 1370 hours into the fermenterrun. Upon the addition of compound D there was a sharp rise in theethanol concentration and acetic acid concentration accompanied by adecrease in the butyrate production. The butanol concentration rosetemporarily following the 100 ppm addition of compound D which again isbelieved to show a rapid conversion of the butyric acid to butanol aswas seen in Example 5.

Example 10

For 12 days after the first injection of compound D into the fermenter,the fermenter was allowed to wash out compound D. Then 500 ppm ofcompound D was injected directly into the fermenter after about 1660hours of fermenter operation. (See FIG. 3) The resulting concentrationsof the C₂ and C₄ alcohols and acids from the time of adding the 500 ppmof compound D are shown in Table 10.

TABLE 10 Addition OD EtOH Acetic acid n-Butanol Butyric acid Hr @ 600 nmg/L g/L g/L g/L 0 1.40 0.55 8.72 0.21 1.51 30 1.35 0.61 8.48 0.23 1.4749 1.23 0.69 8.64 0.26 1.44 73 1.12 0.68 8.56 0.26 1.37 123 0.92 0.538.72 0.19 1.41 145 1.12 0.88 8.75 0.26 1.36 168 1.38 0.95 8.83 0.28 1.11193 1.18 0.87 8.86 0.24 1.12 217 1.24 0.84 9.05 0.21 1.13 240 1.25 0.829.21 0.21 1.17

The data and FIG. 6 show that immediately after addition of the compoundD, the OD began to decrease showing reduction of cell growth.Furthermore, the concentrations of n-butyrate and n-butanol began tolevel off and slowly decrease whereas the concentrations of ethanol andacetate began to rise. Moreover, after the initial decrease, the ODbegan to rise with the concomitant increase of the C₂ compounds. Thisclearly established that the butyrigens were specifically inhibitedwhereas the homoacetogenic organisms were not inhibited and were able togrow and continue to produce the desirable C₂ products in the presenceof compound D.

It is claimed:
 1. A method of restricting the production of butyrate andbutanol in an anaerobic fermentation of a gas substrate comprising atleast one of CO and a mixture of CO₂ with hydrogen, the methodcomprising: passing the gas stream to an anaerobic fermentation zonecontaining at least one species of anaerobic microorganism capable ofproducing a liquid product comprising light oxygenates; converting atleast a portion of the gas stream to the liquid product by contact ofthe microorganism in the fermentation zone with the gas stream; addingat least one of a 4,4,4, tri-halogen substituted 3(methyltri-halogensubstituted) crotonate ester and a 4,4,4, tri-substituted halogen3(methyltri-halogen substituted) crotonic acid to the fermentationliquid in an amount effective to restrict production of butyrate andbutanol; withdrawing fermentation liquid containing the liquid productfrom the fermentation zone; and, recovering the liquid product from thefermentation liquid.
 2. The method of claim 1 wherein the fermentationzone contains a homoacetogenic microorganism for the production of aliquid product comprising ethanol or acetate and is contaminated with abutyrigenic microorganism that produces at least one of butyrate orbutanol.
 3. The method of claim 1 wherein the fermentation zone containsa heteroacetogenic microorganism that produces at least one of aceticacid and ethanol as the liquid product and at least one of butyrate andbutanol.
 4. The method of claim 1 wherein the ethanol containingfermentation liquid contains less than 0.1% butyrate.
 5. The method ofclaim 1 wherein the fermentation zone contains a homoacetogenicmicroorganism that comprises at least one of Clostridiumautoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, andClostridium coskatii and the fermentation zone produces a liquid productcomprising at least one of acetate and ethanol.
 6. The method of claim 1wherein the fermentation zone comprises a suspended cell bioreactor thatsuspends the anaerobic microorganism in the fermentation liquid and theat least one of the 4,4,4, tri-substituted halogen 3(methyltri-halogensubstituted) crotonate ester and the 4,4,4, tri-substituted halogen3(methyltri-halogen substituted) crotonic acid enters the bioreactorwith a media mixture that flows into the fermentation liquid.
 7. Themethod of claim 1 wherein the fermentation zone comprises a membranesupported bioreactor.
 8. The method of claim 1 wherein the at least oneof the 4,4,4, tri-substituted halogen 3(methyltri-halogen substituted)crotonate ester; and the 4,4,4, tri-substituted halogen3(methyltri-halogen substituted) crotonic acid is at least one of Ethyl4,4,4, Trifluoromethyl 3(trifluoromethyl)crotonate; 4,4,4,trifluoro3-(trifluoromethyl)crotonic acid; and Ethyl 4,4,4,trifluoromethyl3-(trichloromethyl) crotonate; 4,4,4, trifluoromethyl3-(trichloromethyl)crotonic acid; Ethyl 4,4,4 trichloromethyl3-(trifluoromethyl)crotonate; 4,4,4 trichloromethyl3-(trifluoromethyl)crotonic acid; Ethyl 4,4,4 trichloromethyl3-(trichloromethyl)crotonate; and 4,4,4 trichloromethyl3-(trichloromethyl)crotonic acid.
 9. The method of claim 1 wherein theanaerobic microorganism is a heteroacetogen that produces liquidproducts having at least 2 to 3 carbon atoms and the liquid productcomprises at least one of acetic acid, ethanol, propanol, and propionicacid.
 10. The method of claim 1 wherein the at least one of the 4,4,4,tri-substituted halogen 3(methyltri-halogen substituted) crotonate esterand the 4,4,4, tri-substituted halogen 3(methyltri-halogen substituted)crotonic acid is added in an amount sufficient to produce aconcentration of at least 10 ppm in the fermentation liquid.
 11. Themethod of claim 1 wherein the at least one of the 4,4,4, tri-substitutedhalogen 3(methyltri-halogen substituted) crotonate ester and the 4,4,4,tri-substituted halogen 3(methyltri-halogen substituted) crotonic acidis added to the fermentation zone as single dose in an amount of atleast 50 ppm.
 12. The method of claim 1 wherein the fermentation zonecomprises a membrane supported bioreactor and the at least one of the4,4,4, tri-substituted halogen 3(methyltri-halogen substituted)crotonate ester and the 4,4,4, tri-substituted halogen3(methyltri-halogen substituted) crotonic acid is added to thefermentation liquid.
 13. A method of restricting the production ofbutyrate and butanol in an anaerobic fermentation of a gas substratecomprising at least one of CO and a mixture of CO₂ with hydrogen, themethod comprising: passing the gas stream to a fermentation zonecontaining more than one species of anaerobic microorganisms and afermentation liquid; converting the gas stream to a liquid productcomprising a light oxygenate by contact of at least one of the anaerobicmicroorganisms in the fermentation zone with the gas stream; adding anamount of at least one of a 4,4,4, tri-substituted halogen3(methyltri-halogen substituted) crotonate ester and a 4,4,4,tri-substituted halogen 3(methyltri-halogen substituted) crotonic acidto the fermentation liquid to inhibit any production of butyrate orbutanol from at least one species of microorganism in the fermentationzone liquid effective to restrict production of butyrate and butanol;withdrawing fermentation liquid containing the liquid product from thefermentation zone; and, recovering the liquid product from thefermentation zone.
 14. The method of claim 13 wherein the ethanolcontaining fermentation liquid contains less than 0.1% butyrate.
 15. Themethod of claim 13 wherein the fermentation zone contains ahomoacetogenic microorganism for the production of a liquid productcomprising ethanol and a butyrigenic microorganism that produces atleast one of butyrate or butanol.
 16. The method of claim 13 wherein theat least one of the 4,4,4, tri-substituted halogen 3(methyltri-halogensubstituted) crotonate ester and the 4,4,4, tri-substituted halogen3(methyltri-halogen substituted) crotonic acid is at least one of Ethyl4,4,4, Trifluoromethyl 3(trifluoromethyl)crotonate; 4,4,4,trifluoro3-(trifluoromethyl) crotonic acid; and Ethyl 4,4,4,trifluoromethyl3-(trichloromethyl)crotonate.
 17. The method of claim 13 wherein theanaerobic microorganism is a heteroacetogen that produces liquidproducts having at least 2 to 3 carbon atoms and the liquid productcomprises at least one of acetic acid, ethanol, propanol, and propionicacid.
 18. The method of claim 13 wherein the at least one of the 4,4,4,tri-substituted halogen 3(methyltri-halogen substituted) crotonate esterand the 4,4,4, tri-substituted halogen 3(methyltri-halogen substituted)crotonic acid is added to the fermentation liquid at a concentration ofat least 10 ppm.
 19. A method of producing a product comprising a C₂oxygenate by the fermentation of a gas substrate comprising at least oneof CO and a mixture of CO₂ with hydrogen using a homoacetogenicmicroorganism to convert the gas stream to the product, the methodcomprising: passing the gas stream to a fermentation zone containing ananaerobic microorganism and a fermentation liquid; converting the gasstream to the product by contact of the anaerobic microorganism in thefermentation zone with the gas stream; adding an amount of at least oneof a 4,4,4, tri-substituted halogen 3(methyltri-halogen substituted)crotonate ester and a 4,4,4, tri-substituted halogen 3(methyltri-halogensubstituted) crotonic acid to the fermentation liquid sufficient torestrict production of butyrate and butanol; withdrawing a productcontaining fermentation liquid from the fermentation zone; and,recovering the product from the fermentation liquid.
 20. The method ofclaim 19 wherein the at least one of the 4,4,4, tri-substituted halogen3(methyltri-halogen substituted) crotonate ester and the 4,4,4,tri-substituted halogen 3(methyltri-halogen substituted) crotonic acidis added in at least one dose in an amount sufficient to produce aconcentration of at least 10 ppm in the fermentation zone.
 21. Themethod of claim 4-19 wherein the at least one of the 4,4,4,tri-substituted halogen 3(methyltri-halogen substituted) crotonate esterand the 4,4,4, tri-substituted halogen 3(methyltri-halogen substituted)crotonic acid is at least one of Ethyl 4,4,4, Trifluoromethyl3(trifluoromethyl)crotonate; 4, 4, 4,trifluoro3-(trifluoromethyl)crotonic acid; Ethyl 4,4,4,trifluoromethyl3-(trichloromethyl)crotonate; 4,4,4, trifluoromethyl3-(trichloromethyl)crotonic acid; Ethyl 4,4,4 trichloromethyl3-(trifluoromethyl)crotonate; 4,4,4 trichloromethyl3-(trifluoromethyl)crotonic acid; Ethyl 4,4,4 trichloromethyl3-(trichloromethyl)crotonate; and 4,4,4 trichloromethyl3-(trichloromethyl)crotonic acid.